Biogenesis of bacterial outer membrane proteins

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Recently, we have devoted considerable effort to determining the role of lipids in the assembly (membrane insertion and folding) of OMPs. This project emerged from our discovery that the assembly of OmpC (an E. coli OMP that forms homotrimers) into lipid vesicles that contain purified BAM (BAM proteoliposomes) requires lipopolysaccharide (LPS). LPS is a unique glycolipid that is found in the outer leaflet of the OM of most Gram-negative bacteria. This was a surprising discovery because the assembly of other OMPs into BAM proteoliposomes is inhibited by LPS. We found that multiple lipid facing basic residues located near the extracellular side of OmpC that are conserved in similar proteins produced by the Enterobacteriales are required for the efficient insertion of OmpC into both BAM proteoliposomes and into the OM in live cells. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency in vivo. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide (LPS). Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to “pull” the protein into the OM when the local environment is perturbed by the lateral opening of the BamA beta barrel during OMP insertion and suggest a surprising role for membrane lipids in the insertion reaction. In a second study we utilized mRNA display to discover novel cyclic peptides that bind to E. coli BamA with high affinity and that might be used as antibiotics. We identified several peptides that arrest the growth of BAM deficient E. coli strains, inhibit OMP assembly in live cells and in vitro, and bind to unique sites within the BamA beta barrel lumen. Remarkably, we also found that if the peptides are added to cultures after a slowly assembling OMP mutant binds to BamA, they accelerate its biogenesis. The experimental data and molecular dynamics simulations strongly suggest that the peptides trap BamA in conformations that block the initiation of OMP assembly but favor a later assembly step during which the BamA beta barrel undergoes a conformational change from an open to a partially closed state. Although the lateral closing of the BamA beta barrel has been shown to be required for the final stages of OMP assembly and the release of OMP clients from BAM, our data provide evidence that the lateral closing of BamA also facilitates the complete insertion of OMPs into the lipid bilayer.